1. Field of the Invention
The present invention relates to the field of fertility and more particularly, to means and methods for determining and diagnosing endometriosis in women.
2. Description of Related Art
Endometriosis is defined as the ectopic presence of endometrial glands and stroma. Endometriotic tissue is comprised of tissue that is histologically similar yet biochemically and functionally different or out of phase from that of the uterine endometrium.
For example, endometriosis differs from its uterine counterpart in steroid responsiveness and receptor content [Vierikko, et al., 1985; Lessey et al., 1989; Melega et al., 1991] and expression of epidermal growth factor and epidermal growth factor receptor [Melega et al., 1991; Haining et a., 1991]. These altered characteristics, combined with an ectopic location, effect the physiological activity of the endometriotic tissue and thereby alter protein synthesis and secretion by the endometriotic tissue. Deviations in protein synthesis and secretion might be useful in developing unique markers for the nonsurgical diagnosis and management of endometriosis. Unfortunately, limited information is available concerning protein synthesis, secretion, regulation and expression in endometriotic tissue.
Applicant has found dissimilarities in protein synthesis and secretory patterns between eutopic and ectopic uterine tissues (endometriotic implants) using a rat model for endometriosis [Sharpe et al., 1991; Sharpe and Vernon, 1993]. Three endometriosis-associated proteins, synthesized and released in an alternate fashion from uterine proteins, were identified. Two endometriotic proteins named ENDO-I and ENDO-II by applicant (Mr 40,000 to 55,000; pI 4.0 to 5.2 and Mr 30,000 to 32,000; pI 7.0 to 9.0, respectively) were produced by endometriotic implants and not the uteri. The third protein (Mr 70,000; pI 5.7), previously identified in uterine explant cultures as progesterone-induced uterine protein-1 (PUP-1) [Sharpe et al., 1991], appeared in endometriotic implant cultures 24-48 hours later than in uterine cultures [Sharpe and Vernon, 1993]. The identities, functions, mechanisms of altered protein synthesis and secretion by the ectopic uterine tissues and their correlation to the human endometriosis condition were not known at that time.
Little information in the literature addresses human endometriotic secretory proteins. Isaacson and coworkers [Isaacson et al., 1989] showed that human endometriotic tissues produce and secrete complement component 3 (C3) in an alternate fashion to that of the uterine endometrium. Secretion of C3 into the peritoneal cavity may elicit some of the immunological phenomena observed in patients with endometriosis and be related to the pathophysiology of the disease. However, while C3 may play a role in the pathophysiology of endometriosis, C3 is also produced by other tissues in the body and therefore is not useful in the development of an endometriosis-specific marker for the disease.
Further identification of biochemical dissimilarities between the uterine endometrium and endometriosis in vitro may enhance understanding of the mechanism(s) of the pathogenicity of the endometriotic tissue in vivo, potentially leading to the development of improved diagnosis and treatment for endometriosis. Therefore, Applicant desired to identify unique proteins synthesized and secreted by human endometriosis and endometrium in vitro and in vivo for the development of endometriosis-specific markers for diagnosis of the disease. Aberrant production or secretion of proteins by ectopic endometrium in the peritoneal cavity may contribute to the processes of endometriosis, infertility, pelvic adhesive disease and pelvic pain. A specific endometriosis-induced protein could be useful in diagnosis and nonsurgical management of the disease.
According to the present invention, a method of diagnosing endometriosis in a female patient suspected of having endometriosis is disclosed. The method includes the steps of obtaining a sample from the patient. The sample can be a fluid sample such as peritoneal fluid or serum. Alternatively tissue samples can be used in the method The sample is analyzed to detect the presence of ENDO-I glycoprotein or its mRNA in the sample compared to non-endometriosis controls who do not express ENDO-I. The protein is characterized by
(i) a molecular weight of 40,000 to 55,000 as determined by two-dimensional SDS-PAGE polyacrylamide gel electrophoresis;
(ii) having an isoelectric point of 4.0 to 5.5; and
(iii) being synthesized and secreted specifically by stromal cells of endometriotic tissue origin; and
(iv) in humans having a cDNA as set forth in SEQ ID No:1.
The sample can be assayed for polypeptides/proteins by immunohistochemical and immunocytochemical staining, ELISA, RIA, immunoblots, Western blotting, immunoprecipitation, functional assays for angiogenesis and macrophage activation using established cell lines as is known in the art. In an alternative embodiment mRNA complementary to the nucleic acid sequence can be assayed by in situ hybridization, Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR).